GABP plays a central role in coordinating expression of numerous respiratory genes, including that of mitochondrial transcription factor 1, which, in turn, regulates mitochondrial gene transcription and DNA replication. Thus, GABP activity directly links nuclear and mitochondrial gene expression. Moreover, the DNA-binding activity of the GABPa subunit is sensitive to cellular redox state, offering a potential mechanism for negative feedback regulation of respiratory gene expression. GABP is composed of two subunits, GABPa and GABPb which form heterodimeric and heterotetrameric complexes bound to DNA containing one or two GABP binding sites, respectively. The aims of the project are two-fold. Firstly, the project will define and characterize the role of GABPa as a redox sensor, through redox changes at three cysteines in the DNA binding and dimerization domains of the GABPa subunit. The research will examine by site-directed mutagenesis and deletion analysis, the functional consequences of sulfhydryl modification of GABPa cysteines on DNA binding and dimerization with GABPb. The physiological impact of mutant GABP proteins expressed in cultured cells will be assessed and may help illuminate the processes of mitochondrial biogenesis and electron transport chain assembly. GABP binds to a single GABP binding site in vitro as a dimer. Transcriptional activation by GABP requires the a2b2 heterotetrameric form of the protein, yet promoters containing a single GABP binding site are known to be regulated by GABP. Clearly, other mechanisms capable of facilitating tetramer assembly on a single PEA3/EBS site must exist. As a second objective, the project will characterize the assembly of GABP dimer and tetramer complexes in the presence and absence of DNA by analytical ultracentrifugation. Regions of GABPa and GABPb proteins involved in regulation of tetramer assembly will be identified through analysis of mutants altered in tetramer complex assembly. Identification of sequences in GABPa and GABPb that facilitate assembly of the GABP hetero-tetramer complex may offer insights into physiological mechanisms (e.g., phosphorylation) for regulating tetramer formation.
Eukaryotic cells generate energy through activities associated with the subcellular organelle known as the mitochondria. The correct regulation of this process is essential for the growth and maintenance of all cells. The research will provide valuable insights into the mechanisms by which mammalian cells coordinate the essential activity of mitochondrial energy production. Through the characterization of the gene regulator, GABP, this research will further the understanding of mitochondrial biogenesis and regulation of cellular energy production.
